The overall goals of the proposed research are to understand from a mechanistic and structure-function perspective how human topoisomerase I manages the topology of DNA in vivo, and how cells repair the damage that occurs when topoisomerase I becomes stalled in covalent complexes on the DNA. Such complexes result from topoisomerase I cleavage in the vicinity of several types of DNA damage and after treatment with the anticancer drug, camptothecin. A consideration of possible repair pathways will include both a detailed analysis of the properties of the enzyme, tyrosyl-DNA phosphodiesterase (Tdp1), and a test of an endonuclease-mediated repair hypothesis. The proposed studies will determine how the linker domain of topoisomerase I regulates the cleavage-religation equilibrium and whether this effect requires a direct interaction between the linker and the substrate DNA. A possible role for the linker in aligning the DNA for blunt-end ligation during illegitimate recombination leading to chromosomal deletions will be investigated. To gain insights into the nature of the in vivo substrates for Tdp1, a series of model structures with modifications in both the DNA and peptide moieties of the substrate will be tested in binding and cleavage assays. A procedure has been developed for the isolation of the topoisomerase I-DNA covalent complexes generated in vivo after exposing cells to camptothecin or expressing a "toxic" topoisomerase I. This procedure will be used to identify repair pathway intermediates that accumulate under conditions of limiting Tdp1. Preliminary results suggesting the possible involvement of an endonuclease in the repair of covalent complexes will also be pursued. To better understand how Tdp1 repairs topoisomerase l-related DNA damage and to investigate the likely possibility that Tdp1 also repairs other types of DNA damage, we will identify and characterize proteins that interact with Tdp1. These studies are medically important because topoisomerase I is the target of a variety of anticancer drugs and because elucidating how Tdp1 or other enzymes repair topoisomerase I-DNA covalent complexes may promote the development of new drugs that could be used in combination anticancer drug therapies. Finally, characterizing the accessory domains of the heterodimeric Leishmania donovani topoisomerase I will likely facilitate the development of new drugs that selectively target this atypical type IB topoisomerase for the treatment of Leishmaniasis.